IRF Luciferase Reporter HEK293 Stable Cell Line

IRF Responsive Luciferase Reporter HEK293 Cell Line is derived from human embryonic kidney, and stably express firefly luciferase reporter gene under the control of IRF response element. This cell line is an ideal cellular model for monitoring the activation of Immune Response Pathway triggered by stimuli treatment, enforced gene expression and gene knockdown.

Principle

Members of the interferon regulatory transcription factor (IRF) family are involved in antiviral defense, cell growth regulation, and immune activation. Initially identified as regulators of type I interferon (IFN) gene induction, IRF family of transcription factors transduce signals for multiple classes of the pattern-recognition receptors (PRRs), such as Toll-like receptors, retinoic acid–inducible gene-I (RIG-I)-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), and other nucleic acid–sensing receptors. When activated, each IRF protein translocates to the nucleus and binds to DNA sequence similar to IFN-stimulated response element (ISRE). IRF activation can enhance the IFN-mediated antiviral immune response. Oppositely, abnormal activation of type I IFNs contributes to the development of autoimmune diseases, such as SLE. Signosis has established an IRF luciferase reporter stable cell line that can be used as a reporter system for monitoring the activation of IRF triggered by stimuli treatment, enforced gene expression and gene knockdown.

The cell line is established by transfection using a pTA-GAS/ISRE-luciferase reporter vector, which contains IRF binding sites, a minimal promoter upstream of the firefly luciferase coding region, along with hygromycin expression vector followed by hygromycin selection. The hygromycin resistant clones were subsequently screened for IFNgamma-induced luciferase activity.

Principle behind TF luciferase reporter. TF luciferase reporter stable cell line utilizes artificial promoter constructs to drive luciferase expression. The promoter region can consists of multiple repeats of a cis-element TF binding site, a DNA fragment from the promoter region of a known TF downstream gene, or a DNA fragment containing putative/known TF binding sites. There are several ways that a TF can be activated, such as through extracellular stimuli or through intracellular signaling pathways. Once activated, the TF translocates to the nucleus and often interacts with relevant co-factors to drive gene expression. Once luciferase is expressed, it can generate light in an enzymatic assay and the amount of light measured is positively correlated with the level of TF activation.

Data

Analysis of IRF Luciferase Reporter HEK293 Stable Cell Line. The HEK293 cells were seeded on a 96-well plate for overnight with DMEM including 10% FBS. The cells then were serum-deprived for 8 hours before treating with the following chemicals in DMEM and 0.1% FBS for 16 hours: 100ng/ml IFN-gamma, 20ng/ml TNFα, 20ng/mL IL-1a, 5ng/mL poly I:C, 10ng/ml PMA, 10ng/mL TGFb, and 50uM TBHQ . IRF Luciferase Reporter HEK293 Cell Line exhibits more than 20-fold response to IFNgamma when compared to untreated cells.